1. Field of the Invention
The present invention relates generally to fluorescent dyes and more particularly to fluorescent dyes useful for the detection and quantitation of a target material in biological assays.
2. Technical Background
Fluorescence labeling is an important technology for detecting biological molecules; for example, antibodies can be labeled with fluorescent dyes. After labeling, the binding of antibodies to their specific target molecules can then be monitored on the basis of a change in fluorescence signal, whether it be an increase or a decrease in fluorescence signal. This change in fluorescence may be detected with a spectrometer, immunofluorescence instrument, flow cytometer, fluorescence microscope, or other detection instrument. In a similar way, DNA sequences can be detected with fluorescence detection instruments after the DNA has been hybridized with a complementary DNA sequence that has been labeled with a fluorescent dye.
Fluorescent dyes offer the opportunity to use color and light to detect and quantify a target material, investigate reactions, and perform assays. Generally, fluorescent dyes with a large Stokes shift (i.e., the difference between fluorescence excitation and emission wavelengths), a low molecular weight and greater stability may permit faster, more sensitive and more selective methods to be utilized.
Commercially available fluorescent dyes have a number of disadvantages. For example, some fluorescent dyes have a quantum yield that is too low and hence lack the sensitivity needed to detect small changes in emission light associated with small amounts of the target material. Other fluorescent dyes have a Stokes shift that is too small to permit detection of emission light without significant detection of excitation light. If the excitation or emission spectrum of the fluorescent dye overlaps with the auto fluorescence of the target material causing a low signal-to-noise ratio, the target material is undetectable. Further, some fluorescent dyes are not stable, having a short shelf-life, so that they are readily bleached and rendered nonfluorescent. Still other fluorescent dyes have an excitation spectrum that does not permit them to be excited by wavelength-limited light sources, such as common lasers and arc lamps. In addition to the above disadvantages, many commercially available fluorescent dyes have the further disadvantage in that they are insoluble in aqueous media. Consequently, they must be dissolved in organic solvents, for example, N,N-Dimethylformamide (DMF) prior to substrate labeling in aqueous media, and these organic solvents can have a deleterious effect upon sensitive substrates. The solubility of the fluorescent dye affects the degree to which they interact with themselves in solution and, when conjugated to substrates, directly influences their light absorption and emission properties. The degree of non-specific staining of cellular matter by a fluorescent dye, which increases noise in the signal-to-noise ratio during fluorescent measurement, is also a function of the dye's hydrophobicity and of the polarity of the fluorescent dye's appended functional groups. Lastly, many fluorescent dyes are difficult to synthesize and expensive to purchase, leading to high-cost detection methods, for example, biological assays for DNA, antigen, monoclonal antibodies, and other assays known in the art.
Although the bases of DNA exhibit an absorbance in the UV spectrum at 260 nm, quantification of DNA by this method requires a large amount of material due to the low sensitivity (high extinction coefficient). It is often quite difficult and costly to obtain sufficient material needed for absorbance detection of DNA alone. As the pharmaceutical and biotech industries continue the trend toward smaller scale and higher throughput assays, more sensitive means of nucleic acid detection and quantitation are required. Assays based on fluorescence are attractive due to their high selectivity and sensitivity, thus allowing for the use of less material.
Cyanine dyes, which exhibit a change in fluorescence intensity upon binding to DNA, are the industry workhorse for biological applications. Cyanine dyes have been developed for nucleic acid detection that are very sensitive over large dynamic ranges. An exemplary cyanine dye is PicoGreen™ (a trademark of Molecular Probes, Inc.) which has a sensitivity for double-stranded DNA (dsDNA) of 250 pg/mL on a microplate reader and a dynamic range ˜3 orders of magnitude. PicoGreen™, while not specific for dsDNA over single-stranded DNA (ssDNA), can quantitate dsDNA in the presence of equimolar ssDNA. In addition, it has also been shown that PicoGreen™ can differentiate dsDNA and ssDNA based on its fluorescence lifetime, a sophisticated and cumbersome experiment. Thus, while commercially available cyanine ayes can differentiate between dsDNA and ssDNA, they are not completely satisfactory due to their high cost and small Stokes shifts (typically less than 20 nm). A small Stokes shift causes difficulty in reading the emission signal over the noise in the assay. To compensate for a small Stokes shift, it is necessary to use narrow wavelength laser excitation sources and multiple emission high resolution monochromators, which ultimately results in increased instrument cost.
As a result of the foregoing problems with currently available commercial dyes, fluorescent dyes which can be easily synthesized from relatively inexpensive starting materials, which exhibit a large Stokes shift and which have a low molecular weight are desirable. These properties will permit cleaner detection of the dye emission wavelength over the excitation wavelength, thus eliminating the need for more expensive instrumentation. Fluorescent dyes which have an increased selectivity towards dsDNA over ssDNA, a feature which is important when quantifying dsDNA in a crude reaction mixture (i.e. PCR product mix), are also desirable. Fluorescent dyes which have multiple attachment sites for target material to bind, tethering ability from the multiple attachment sites for attaching the fluorescent dye to a surface, fluorescence in the dry state (requiring no solvent), and a long shelf-life would also be beneficial. Fluorescent dyes soluble in aqueous media which can be manipulated based on the particular application would be especially advantageous.